This Phase II proposal aims to develop and commercialize IR+Raman a breakthrough instrument that will for the first time enable simultaneous infrared (IR) and Raman spectroscopy on the same instrument with sub-micron spatial resolution. This project is well aligned with NIH goals as it incorporates several key thrusts of the National Institute of Biomedical Imaging and Bioengineering, including optical imaging and spectroscopy, infrared imaging, confocal microscopy, and multimodal imaging. IR and Raman have gained interest in investigating the composition and molecular structure of biological materials as they operate label free and are sensitive towards macromolecular composition, such as proteins, lipids, nucleic acids and carbohydrates, as well as the detection of isotopic labelling of these macromolecules and even smaller metabolites. Both Infrared and Raman spectroscopies are widely used in analytical laboratories and are often referred to as ?complementary techniques? as they both probe different types of molecular vibrations. For example, IR spectroscopy is very sensitive to protein secondary structure, whereas Raman is particularly sensitive to lipids as well as certain amino acids. And in pharma applications Raman is more sensitive to drugs, whereas IR is more sensitive to excipients (additives) that often have weak Raman signals and/or have large fluorescent backgrounds. Raman can achieve sub-micron spatial resolution, but IR is limited by the longer excitation wavelengths to spatial resolution ~10 um. This project aims to overcome this limitation by providing IR and Raman spectroscopy, both at sub-micron spatial resolution. A compelling recent example of the power of the multimodal combination of IR and Raman in health sciences involved analysis of malaria parasite infected red blood cells (D. Perez-Guaita et al Vib. Spectrosc. 91, 46-58 (2017)). The research showed ?that the combination of both techniques provides complementary information not evident? using the techniques individually. This research was performed however using a painstaking process of separately and sequentially measuring the exact same cell locations in two different instruments, requiring substantial additional time and cost. This proposal aims to develop an instrument that makes simultaneous IR and Raman measurements simple, robust, and routine. This project will leverage successful Phase I research to develop and commercialize a new optical microscope-based platform that can perform simultaneous IR and Raman on the same instrument. The project will involve a collaboration between proposer Photothermal Spectroscopy Corp and Dr. Ji-Xin Cheng (Boston University) and Dr. Lynne Taylor (Purdue). The team at photothermal will design and build a next generation IR+Raman instrument to overcome key limitations and expand the capabilities over the prototype developed in Phase I. The two year project will develop alpha and beta prototype units for applications testing at Photothermal?s applications lab in Santa Barbara, CA, and will install a beta unit in the labs of Prof. Lynne Taylor at Purdue University, with a focus on demonstrating applicability of IR+Raman to key problems in pharmaceutical sciences. Photothermal scientists will also collaborate closely with Prof. Cheng?s group at Boston University in cell biology, specifically related to investigate antibody susceptibility at the single bacterium level. The beta IR+Raman will also be used to investigate other applications in cells/tissue and microplastics characterization.